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Individuals living with type 1 diabetes mellitus may experience an increased risk of long bone fracture. These fractures are often slow to heal, resulting in delayed reunion or non-union. It is reasonable to theorize that the underlying cause of these diabetes-associated osteopathies is faulty repair dynamics as a result of compromised bone marrow progenitor cell function. Here it was hypothesized that the administration of non-diabetic, human adult bone marrow-derived mesenchymal stromal cells (MSCs) would enhance diabetic fracture healing. Human MSCs were locally introduced to femur fractures in streptozotocin-induced diabetic mice, and the quality of de novo bone was assessed eight weeks later. Biodistribution analysis demonstrated that the cells remained in situ for three days following administration. Bone bridging was evident in all animals. However, a large reparative callus was retained, indicating non-union. &micro, CT analysis elucidated comparable callus dimensions, bone mineral density, bone volume/total volume, and volume of mature bone in all groups that received cells as compared to the saline-treated controls. Four-point bending evaluation of flexural strength, flexural modulus, and total energy to re-fracture did not indicate a statistically significant change as a result of cellular administration. An ex vivo lymphocytic proliferation recall assay indicated that the xenogeneic administration of human cells did not result in an immune response by the murine recipient. Due to this dataset, the administration of non-diabetic bone marrow-derived MSCs did not support fracture healing in this pilot study.

To investigate the feasibility of tooth regeneration by seeding cranial neural crest stem cell (CNCSC) in vivo.Cranial neural tubes, dissected from mouse E9 d, were explanted onto fibronectin-coated dishes. CNCSC emigrated from the explanted neural tubes, and were cultured in a free-serum medium containing modified DMEM/F12. CNCSC, induced by FGF8, BMP2, TGFbeta1 and dentin matrix non-collagen protein (DMNCP), were cultured with collagen/chitosan, and implanted into the subcutaneous part of immunodeficiency mouse. The expression of collagen I/dentin sialophosphoprotein (DSPP) was analyzed by immunocytochemistry.With the scaffolds destroying, columnar cells possessing polarized nuclei and matrix produced by cells were showed in some regions. Immunohistochemical staining demonstrated that collagen type I and DSPP were expressed throughout the cytoplasm and matrix produced by cells.By tissue engineering approach, our experiments further verify the odontoblast-like cell phenotype differentiation of CNCSC in vivo.

To investigate the feasibility of repairing bone defect with methods of tissue-engineering and human bone morphogenetic protein-2 (hBMP-2) gene transfection in osteoporotic rats.Twenty-four 6-month-old female Sprague-Dawley rats underwent ovariectomy, while 8 rats received sham-operations. Three months later, bone mesenchymal stem cells (BMSC) harvested from osteoporotic rats were divided into two groups randomly. Experimental group were transfected by recombinant plasmid carrying hBMP-2 gene, and control group left untreated. All BMSC were seeded into coralhydroxyapatite scaffolds. Then the cell/scaffold constructs were implanted into the defect site created in the ramus of mandible of osteoporotic rats respectively.Positive results were confirmed by immunohistochemistry and in situ hybridization in experimental group. New bone formation was found at the margin of the defect treated with the BMSC modified by hBMP-2 gene transfer at 4 weeks after implantation and appeared mature 8 weeks after the treatment. However, the amount of newly formed bone was much less and there was some adipose tissue at defect margins 8 weeks after implantation in control group.The results of this experiment indicate that BMSC-mediated rhBMP-2 gene therapy in conjunction with bone tissue engineering may allow for successful treatment of large bone defects in osteoporosis rats.

To make a comparison on the efficiency of two methods for transfecting Green Fluorescence Protein gene into human adipose tissue-derived stromal cells, and to study the biological properties and multipotential differentiation of gene-transfected cells.The human subcutaneous adipose tissue was obtained, digested with one volume of collagenase type I, and then cultured with BGJb medium. After subculture and expansion, the human adipose tissue-derived stromal cells infected with Ad-GFP or liposome were observed and analyzed with fluorescence microscopy and flow cytometry to assess transfection efficiency. The growth curve of transfected adipose tissue-derived stromal cells was protracted. The adipose tissue-derived storomal cells were induced to differentiate into osteoblasts, and non-transfected cells were set as control.42.5% +/- 1.5 of the human adipose tissue-derived stomal cells infected with Ad-GFP were found to express GFP at a level higher than that of the control of liposome (11.40%). Infected adipose tissue-derived stromal cells were noted to form mineralized nodes by the use of Alizarin Red stain.The human adipose tissue-derived stromal cells infected with Ad-GFP can express higher level of GFP, and can maintain the ability of proliferation and differentiation as the non-infected human adipose tissue-derived stromal cells do. The infected adipose tissue-derived stromal cells with Ad-GFP can track the change of adipose tissue-derived stromal cells in the study of multipotential differentiation and can serve as cellular vehicles for systemic gene delivery.

The purpose of this study was to evaluate the feasibility of VEGF165 gene transfection into bone marrow stromal cells and make the groundwork of VEGF gene therapy for the revascularization of bone tissue engineering.Bone marrow stromal cells (BMSC) were cultured in vitro, and were tested for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. The vector pcDNA3.1-VEGF165 was transfected into bone marrow stroma cells with the method of liposome mediated or Sofast transfectam reagent. The expression level of human VEGF165 in the transformed cells was detected by immunocytochemistry.The percent of the positive cells after VEGF165 gene transfection mediated by liposome and cationic polymer was 11.34 and 11.42 respectively. There was no significant difference in gene transfer efficacy between liposome and cationic polymer mediated gene delivery. While the percent of the number of survival cells after gene transfection was 74.60 and 85.88 respectively, the cytoxity of liposome was a little higher than that of cationic polymer.The experiment demonstrated that rhVEGF165 was successfully expressed in BMSC. This result could serve as a basis for the next step of revascularization of bone tissue engineering.

To study the multi-lineage potential of bone marrow mesenchymal stem cells (MSCs) derived from transgenic mice with green fluorescent protein (GFP) gene in vitro.A 6-week-old GFP transgenic mouse was executed by dislocation of cervical vertebra, and the marrow in tibia and thighbone was washed out with asepsis. The limited cell strains of MSCs derived from GFP transgenic mice (GFP-MSCs) were obtained with density gradient centrifugation. The passage 3 GFP-MSCs were induced to differentiate into osteoblast, adippcyte, neuron with solution of calcium induction medium, adipogenic medium and neural induction medium respectively. After being calcium-induced, the activity of alkaline phosphatase on GFP-MSCs was determined by micro-plate reader, and alizarin red staining was performed to test the formation of calcium concentration. The adipo-induced MSCs were detected with oil red O staining. Immunocytochemical staining was performed to detect the expression of NSE on neuron-induced MSCs.The ALP activity of GFP-MSCs heightened gradually along with being calcium-induced, and alizarin red staining showed positive. Oil red O staining of adipo-induced cells and NSE immunocytochemical staining of neuron-induced cells demonstrated positive.The limited cell strain of GFP-MSCs possesses multi-lineage potential, which can be used as an efficient tracking facility for studying the mechanism of multi-lineage potential on the MSCs.

To study the biological features and osteoblast/adipocyte phenotypes of bone marrow stromal cells (BMSCs) of Sprague-Dawley (SD) rats with Type I osteoporosis by induced culture.Six-month-old SD rats were used in this study. 16 female rats were randomly divided into two groups. Eight rats were ovariectomied as experimental group to establish the modle of Type I osteoporosis, while other rats received sham-operation. Three month later BMSCs of 16 rats were isolated by discontinueous gradient centrifugation and then plated in alpha-MEM medium as primary culture. Secondary harvested cells were cultured for 14 days in alpha-MEM medium supplemented with dexamethasone, ascorbic acid, vitamin D3, beta-glycerophosphate or dexamethasone, 3-isobutyl-1-methylxanthine, insuline, and indomethine. The cells were screened by inverted microscope each day and cell growth was studied with cell counting. The osteoblast and adipocyte phenotypes were verified by cytochemistry staining, counted the percentage of positive stained cells.The weight and bone mineral density of rats were statistically different between experimental group and control group. Gomori and Von Kossa's staining demonstrated positive osteoblast phenotypes of alkaline phosphatase and mineralized nods by osteogenic inducer, while Oil Red O staining identified BMSCs treated with adipogenic medium resulted in adipocyte formation and there was no significant difference in the percentage of positive stained cells between two groups.The model of Type I osteoporosis has been established successfully. BMSCs from SD rats with osteoporosis maintain their differentiation potential.

To investigate the chondrogenisis by alginate gelatin and rats' bone marrow stromal cells (BMSCs) chondrogenicly induced in vitro.Thirty-two male adult SD rats were assigned randomly to experimental and control groups. In experimental group, bone marrow was obtained from the right tibias of all the rats. After expanding and culturing 3 passages, induced BMSCs by chondrogenic culture medium for 10 days. Suspended induced cells in alginate gelatin, and injected the complex into the hypodermic tissue of the backs of rats autogenously. In control group only alginate gelatins were injected. The grafts were taken out for examinations 4 and 8 weeks after the operations.Considerable cartilage appeared in experimental group 8 weeks after operations. Regular HE staining and alcian blue staining showed a great deal of cartilage holding chondrocyte masses surrounded by abundant matrix. Alginate gelatin decompounded obviously, and the rest distributed among newly formed cartilage. No cartilage appeared in control group all through.BMSCs and alginate gelatin have a beautiful future in cartilage tissue engineering.

To isolate human adipose tissue-derived stromal cells and study the potential of osteogenic differentiation after inductive culture.Liposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were plated in BGJb medium as primary culture for ten days. The second passage cells were harvested and plated in DMEM/F12 medium supplemented with 10% FBS, 5% horse serum and 50 micromol/L hydrocortisone for myogenic induction culture. The cell-anchored slips were removed and fixed in 4% formaldehydam polymerisatum. Toluidine blue, Mallory's phosphotungstic hematoxylin staining and monoclonal antibody to human skeletal muscle myosin heavy chain immunocytochemical methods were used to assay the differentiation of cells.It was observed that the size and shape of induced cells were much different from those of non-induced cells. Toluidine blue, Mallory's phosphotungstic hematoxylin staining demonstrated there were many basophilic striations within cytoplasm and multinucleated myotubes were formed. Immunocytochemical stain indicated that characterastic skeletal myosin heavy chain was positive in myogenic induced cells.It seems that human adipose tissue represents an abundant reservoir of adult stem cells that have multi-germline potential to differentiate into myoblasts. Adipose tissue derived stromal cells will be another alternative source for cell-based tissue engineering in skeletal muscle reconstruction.

To isolate and chondro-inductive culture of human adipose tissue-derived stromal cells and to study their heterotopic chondrogenesis by loading them on alginate gel.Liposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were primarily cultured in BGJb medium for ten days. Secondary harvested cells were cultured in DMEM-F12 medium supplemented with 10%FBS, 6.25 mg/L insulin, 10 mg/L TGF-beta1, 50 mg/L of freshly prepared L-ascorbate for 14 days. After in vitro assay of chondrogenic phenotypes, the cells at density of 10(10)/L were mixed with 1.2% alginate sodium and 102 mmol/L CaCl(2). The cross-linking cell-alginate gel were injected into four BALB/C athymic mice subcutaneously (1 ml for each mouse). Meanwhile, the auto-controls were set by injecting equal dose of simple alginate gel and pure cells in two opposite buttocks of the same mouse subcutaneously. Two mice were sacrificed at fourth and eighth week postoperatively and all samples were removed, fixed, embedded in paraffin and cut into sections of 5 micro m thick. HE staining, Alcian blue and modified Masson's trichrome staining were employed to observe chondrogenesis histologically.Alcian blue and immunocytochemical staining revealed chondroitin sulfate and collagen II in cell matrix after having been chondro-inductive cultured for 14 days. At intervals of fourth and eighth week, heterotopic chondrogenesis is (cartilage formed) within cell-alginate injected sites were found in all mice but negatively in auto-controls. Histologically the hypertrophic chondrocytes were among cartilage matrix in different staining. All alginate gel and solitory cells absorbed within two to three weeks postoperatively in auto-controls.It seems that stromal cells derived from human adipose tissue presents a potential for chondrogenic differentiation.

To establish a new culture system for mouse tooth germs in chick embryo.The mandibular first molar germ fragments of 15 embryonic days' Kunming mouse embryo were implanted into the lateral mesenchyme of 4-5 days' chick embryo wing buds in ove. Eggs were reincubated and implanted tissues were examined by histochemistry.The cultured tooth germ development continued from cap stage to latest bell stage. The ameloblast and the odontoblast all differentiated maturely and secreted matrix.4-5 days' wing buds chick embryo could serve as developing the mouse tooth germs and demonstrate well physiological process of differentiation and morphogenesis.

To construct the eukaryotic expression vector containing human vascular endothelial growth factor 165(VEGF165) gene and express it in rat bone marrow stroma cells(rMSCs).The recombinant plasmid pSP73-VEGF165 was digested with BamH I and Xho I. Then the hVEGF165 gene segment obtained was again cloned into pcDNA3.1 to construct recombinant eukaryotic expression vector pcDNA3.1-VEGF165. Then the recombinant vector was identified by enzyme digestion analysis and sequencing. The rMSCs were transformed by recombinant vector and positive clones were screened with G418. The expression of hVEGF165 gene in the transformed cells was detected by immunocytochemical staining.Enzyme digestion analysis and sequencing showed that target gene had been cloned into recombinant vector. The expression of hVEGF165 gene in the transformed cells had been demonstrated by immunocytochemical staining.The recombinant eukaryotic expression vector has been constructed and expressed successfully in the transformed cells. Therefore, it is possible to use the rMSCs expressing hVEGF165 gene as seed cells in the bone tissue-engineering.